The present invention relates to a strategy and medicaments for suppressing a gene. In particular the invention relates to the suppression of mutated genes which give rise to a dominant or deleterious effect either monogenically or polygenically. The invention relates to a strategy for suppressing a gene or disease allele such that (if required) a replacement gene, gene product or alternative gene therapy can be introduced.
The invention also relates to a medicament or medicaments for use in suppressing a gene or disease allele which is present in a genome of one or more individuals or animals. The said medicament(s) may also introduce the replacement gene sequence, product or alternative therapy.
Generally the strategy of the present invention will be useful where the gene, which is naturally present in the genome of a patient, contributes to a disease state. Generally, the gene in question will be mutated, that is, will possess alterations in its nucleotide sequence that affect the function or level of the gene product. For example, the alteration may result in an altered protein product from the wild type gene or altered control of transcription and processing. Inheritance or the somatic acquisition of such a mutation can give rise to a disease phenotype or can predispose an individual to a disease phenotype. However the gene of interest could also be of wild type phenotype, but contribute to a disease state in another way such that the suppression of the gene would alleviate or improve the disease state.
Studies of degenerative hereditary ocular conditions, including Retinitis Pigmentosa (RP) and various macular dystrophies have resulted in a substantial elucidation of the molecular basis of these debilitating human eye disorders. In a collaborative study, applying the approach of genetic linkage, two x-linked RP genes were localised to the short arm of the X chromosome (Ott et al. 1990). In autosomal dominant forms of RP (adRP) three genes have been localised. The first adRP gene mapped on 3q close to the gene encoding the photoreceptor specific protein rhodopsin (McWilliam et al. 1989; Dryja et al. 1990). Similarly, an adRP gene was placed on 6p close to the gene encoding the photoreceptor specific protein peripherin/RDS (Farrar et al. 1991a,b; Kajiwara et al. 1991). A third adRP gene mapped to 7q (Jordan et al. 1993); no known candidate genes for RP reside in this region of 7q. In addition, the disease gene segregating in ha Best""s macular dystrophy family was placed on llq close to the region previously shown to be involved in some forms of this dystrophy (Mansergh et al. 1995). Recently, an autosomal recessive RP gene was placed on 1q (Van Soest et al. 1994). Genetic linkage, in combination with techniques for rapid mutational screening of candidate genes, enabled subsequent identification of causative mutations in the genes encoding rhodopsin and peripherin/RDS proteins. Globally about 100 rhodopsin mutations have now been found in patients with RP or congenital stationary night blindness. Similarly about 40 mutations have been characterised in the peripherin/RDS gene in patients with RP or with various macular dystrophies.
Knowledge of the molecular aetiology of some forms of human inherited retinopathies has stimulated the establishment of methodologies to generate animal models for these diseases and to explore methods of therapeutic intervention; the goal being the development of treatments for human retinal diseases (Farrar et al. 1995). Surgical procedures enabling the injection of sub-microlitre volumes of fluid intravitinally or sub-retinally into mouse eyes have been developed by Dr. Paul Kenna. In conjunction with the generation of animal models, optimal systems for delivery of gene therapies to retinal tissues using viral (inter alia Adenovirus, Adeno Associated Virus, Herpes Simplex Type 1 Virus) and non-viral (inter alia liposomes, dendrimers) vectors alone or in association with derivatives to aid gene transfer are being investigated.
Generally, gene therapies utilising both viral and non-viral delivery systems have been applied in the treatment of a number of inherited disorders; of cancers and of some infectious disorders. The majority of this work has been undertaken on animal models, although, some human gene therapies have been approved. Many studies have focused on recessively inherited disorders, the rationale being, that the introduction and efficient expression of the wild type gene may be sufficient to result in a prevention/amelioration of disease phenotype. In contrast gene therapy for dominant disorders will require the suppression of the dominant disease allele. Notably the majority of characterised mutations that cause inherited retinal degenerations such as RP are inherited in an autosomal dominant fashion. Indeed there are over 1,000 autosomal dominantly inherited disorders in man. In addition there are many polygenic disorders due to the co-inheritance of a number of genetic components which together give rise to a disease phenotype. Effective gene therapy in dominant or polygenic disease will require suppression of the disease allele while in many cases still maintaining the function of the normal allele.
Strategies to differentiate between normal and disease alleles and to selectively switch off the disease allele using suppression effectors inter alia antisense DNA/RNA, ribozymes or triple helix DNA, targeted towards the disease mutation may be difficult in many cases and impossible in othersxe2x80x94frequently the disease and normal alleles may differ by only a single nucleotide. For example, the disease mutation may not occur at a ribozyme cleavage site. Similarly the disease allele may be difficult to target specifically by antisense DNA/RNA or triple helix DNA if there are only small sequence differences between the disease and normal alleles. A further difficulty inhibiting the development of gene therapies is the heterogeneous nature of some dominant disordersxe2x80x94many different mutations in the same gene give rise to a similar disease phenotype. The development of specific gene therapies for each of these would be extremely costly. To circumvent the dual difficulties associated with specifically targeting the disease mutation and the genetic heterogeneity present in some inherited disorders, the present invention aims to provide a novel strategy for gene suppression and replacement exploiting the noncoding and control regions of a gene.
Suppression effectors have been used previously to achieve specific suppression of gene expression. Antisense DNA and RNA has been used to inhibit gene expression in many instances. Many modifications, such as phosphorothioates, have been made to antisense oligonucleotides to increase resistance to nuclease degradation, binding affinity and uptake (Cazenave et al. 1989; Sun et al. 1989; McKay et al. 1996; Wei et al. 1996). In some instances, using antisense and ribozyme suppression stategies has led to the reversal of the tumor phenotype by greatly reducing the expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine 1993; Lange et al. 1993; Valera et al. 1994; Dosaka-Akita et al. 1995; Feng et al. 1995; Quattrone et al. 1995; Ohta et al. 1996). For example, neoplastic reversion was obtained using a ribozyme targeted to the codon 12 H-ras mutation in bladder carcinoma cells (Feng et al. 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeting trans-splicing (Sullenger and Cech 1994; Jones et al. 1996). Ribozymes can be designed to elict autocatalytic cleavage of RNA targets. However the inhibitory effect of some ribozymes may be due in part to an antisense effect of the variable antisense sequences flanking the catalytic core which specify the target site (Ellis and Rodgers 1993; Jankowsky and Schwenzer 1996). Ribozyme activity may be augmented by the use of non-specific nucleic acid binding protiens or facilitator oligonucleotides (Herschlag et al. 1994; Jankowsky and Schwenzer 1996). Triple helix approaches have also been investigated for sequence specific gene suppressionxe2x80x94triplex forming oligonucleotides have been found in some cases to bind in a sequence specific manner (Postel et al. 1991; Duval-Valentin et al. 1992; Hardenbol and Van Dyke 1996; Porumb et al. 1996). Similarly peptide nucleic acids have been shown in some instances to inhibit gene expression (Hanvey et al. 1992; Knudson and Nielson 1996). Minor groove binding polyamides have been shown to bind in a sequence specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al. 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz 1987; Rimsky et al. 1989; Wright et al. 1989). In some cases suppression strategies have lead to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA levels have been mirrored by reductions in protein levels.
The present invention aims to circumvent the shortcomings in the prior art by using a two step approach for suppression and replacement.
According to the present invention there is provided a strategy for suppressing expression of an endogenous gene, wherein said strategy comprises providing suppression effectors able to bind to the non-coding regions of a gene to be suppressed, to prevent the functional expression thereof. Preferably the suppression effectors are antisense nucleic acids. Preferably the targetted non-coding regions include the transcribed but non-translated regions of a gene.
Generally the term suppression effectors includes nucleic acids, peptide nucleic acids (PNAs) or peptides which can be used to silence or reduce gene expression in a sequence specific manner.
The antisense nucleic acids can be DNA or RNA, can be directed to 5xe2x80x2 and/or 3xe2x80x2 untranslated regions and/or to introns and/or to control regions or to any combination of such untranslated regions. However targetted the binding of the antisense nucleic acid prevents or lowers the functional expression of the endogenous gene. Chimeric antisense nucleic acids including a small proportion of translated regions of a gene can be used in some cases to help to optimise suppression. Likewise chimeric antisense nucleic acids including a small proportion of promoter regions of a gene can be used in some cases to help to optimise suppression.
Generally the term xe2x80x98functional expressionxe2x80x99 means the expression of a gene product able to function in a manner equivalent to or better than a wild type product. In the case of a mutant gene xe2x80x98functional expressionxe2x80x99 means the expression of a gene product whose presence gives rise to a deleterious effect.
In a particular embodiment of the invention the strategy further employs ribozymes. These can be designed to elicit cleavage of target RNAs.
The strategy further employs nucleotides which form triple helix DNA.
Nucleic acids for antisense, ribozymes and triple helix may be modified to increase stability, binding efficiencies and uptake as discussed earlier. Nucleic acids can be incorporated into a vector. Vectors include DNA plasmid vectors, RNA or DNA virus vectors. These can be combined with lipids, polymers or other derivatives to aid gene delivery and expression.
The invention further provides the use of antisense nucleotides, ribozymes, triple helix nucleotides or other suppression effectors alone or in a vector or vectors, wherein the nucleic acids are able to bind specifically to untranslated regions of a gene such as the 5xe2x80x2 and 3xe2x80x2 UTRs to prevent the functional expression thereof, in the preparation of a medicament for the treatment of an autosomal dominant disease.
In a further embodiment the non-coding regions of the gene can include promoter regions which are untranslated.
According to the present invention there is provided a strategy for suppressing an endogenous gene and introducing a replacement gene, said strategy comprising the steps of:
1. providing antisense nucleic acid able to bind to at least one non-coding or untranslated region of a gene to be suppressed and
2. providing genomic DNA or cDNA encoding a replacement gene sequence,
wherein the antisense nucleic acid is unable to bind to equivalent non-coding or untranslated regions in the genomic DNA or cDNA to prevent expression of the replacement gene sequence.
The replacement nucleic acids will not be recognised by the suppression nucleic acid. The control sequences of the replacement nucleic acid may belong to a different mammalian species, may belong to a different human gene or may be similar but altered from those in the gene to be suppressed and may thus permit translation of the part of the replacement nucleic acid to be initiated.
By control sequences is meant sequences which are involved in the control of gene expression or in the control of processing and/or sequences present in mature RNA transcripts and/or in precursor RNA transcripts, but not including protein coding sequences.
In a particular embodiment of the invention there is provided a strategy for gene suppression targeted towards the non-coding regions of a gene and using a characteristic of one of the alleles of a gene, for example, the allele carrying a disease mutation. Suppressors are targeted to non-coding regions of a gene and to a characteristic of one allele of a gene such that suppression in specific or partially specific to one allele of the gene. The invention further provides for replacement nucleic acids containing altered non-coding sequences such that replacement nucleic acids cannot be recognised by suppressors which are targeted towards the non-coding regions of a gene. Replacement nucleic acids provide the wild type or an equivalent gene product but are protected completely or in part from suppression effectors targeted to non-coding regions.
In a further embodiment of the invention there is provided replacement nucleic acids with altered non-coding sequences such that replacement nucleic acids cannot be recognised by naturally occurring endogenous suppressors present in one or more individuals, animals or plants. Replacement nucleic acids with altered non-coding sequences provide the wild type or equivalent gene product but are completely or partially protected from suppression by naturally occurring endogenous suppression effectors.
In an additional embodiment of the invention there is provided replacement nucleic acids with altered non-coding sequences such that replacement nucleic acids provide a wild type or equivalent gene product or gene product with beneficial characteristics. For example, the 3xc2x0 non-coding sequences of the replacement nucleic acids could be altered to modify the stability and turn over the RNA expressed from the replacement nucleic acids thereby sometimes affecting levels of resulting gene product.
The invention further provides the use of a vector or vectors containing suppression effectors in the form of nucleic acids, said nucleic acids being directed towards untranslated regions or control sequences of the target gene and vector(s) containing genomic DNA or cDNA encoding a replacement gene sequence to which nucleic acids for suppression are unable to bind, in the preparation of a combined medicament for the treatment of an autosomal dominant disease. Nucleic acids for suppression or replacement gene nucleic acids may be provided in the same vector or in separate vectors. Nucleic acids for suppression or replacement gene nucleic acids may be provided as a combination of nucleic acids alone or in vectors. The vector may contain antisense nucleic acid with or without, ribozymes.
The invention further provides a method of treatment for a disease caused by an endogenous mutant gene, said method comprising sequential or concomitant introduction of (a) antisense nucleic acids to the non-coding regions of a gene to be suppressed; to the 5xe2x80x2 and/or 3xe2x80x2 untranslated regions of a gene or intronic regions or to the non-control regions of a gene to be suppressed, (b) replacement gene sequence with control sequences which allow it to be expressed.
The nucleic acid for gene suppression can be administered before or after or at the same time as the replacement gene is administered.
The invention further provides a kit for use in the treatment of a disease caused by an endogenous mutation in a gene, the kit comprising nucleic acids for suppression able to bind to the 5xe2x80x2 and/or 3xe2x80x2 untranslated regions or intronic regions or control regions of the gene to be suppressed and (preferably packaged separately thereto) a replacement nucleic acid to replace the mutant gene having a control sequence to allow it to be expressed.
Nucleotides can be administered as naked DNA or RNA, with or without ribozymes and/or with dendrimers. Ribozymes stabilise DNA and block transcription. Dendrimers (for example dendrimers of methylmethacrylate) can be utilised, it is believed the dendrimers mimic histones and as such are capable of transporting nucleic acids into cells. Oligonucleotides can be synthesized, purified and modified with phosphorothioate linkages and 2xe2x80x20-allyl groups to render them resistant to cellular nucleases while still supporting RNase H medicated degradation of RNA. Also, nucleic acids can be mixed with lipids to increase efficiency of delivery to somatic tissues.
Nucleotides can be delivered in vectors. Naked nucleic acids or nucleic acids in vectors can be delivered with lipids or other derivatives which aid gene delivery. Nucleotides may be modified to render them more stable, for example, resistant to cellular nucleases while still supporting RNaseH mediated degradation of RNA or with increased binding efficiencies as discussed earlier.
Suppression effectors and replacement sequences can be injected sub-sectionally, or may be administered systemically.
There is now an armament with which to obtain gene suppression. This, in conjunction with a better understanding of the molecular etiology of disease, results in an ever increasing number of disease targets for therapies based on suppression. In many cases, complete (100%) suppression of gene expression has been difficult to achieve. Possibly a combined approach using a number of suppression effectors may be required. For some disorders it may be necessary to block expression of a disease allele completely to prevent disease symptoms whereas for others low levels of mutant protein may be tolerated. In parallel with an increased knowledge of the molecular defects causing disease has been the realisation that many disorders are genetically heterogeneous. Examples in which multiple genes and/or multiple mutations within a gene can give rise to a similar disease phenotype include osteogenesis imperfecta, familial hypercholesteraemia, retinitis pigmentosa, and many others.
The invention addresses some shortcomings of the prior art and aims to provide a novel approach to the design of suppression effectors directed to target mutant genes. Suppression of every mutation giving rise to a disease phenotype may be costly, problematic and sometimes impossible. Disease mutuations are often single nucleotide changes. As a result differentiating between the disease and normal alleles may be difficult. Furthermore some suppression effectors require specific sequence targets, for example, ribozymes can only cleave at NUX sites and hence will not be able to target some mutations. Notably, the wide spectrum of mutations observed in many diseases adds an additional layer of complexity in the development of therapeutic strategies for such disorders. A further problem associated with suppression is the high level of homology present in coding sequences between members of some gene families. This can limit the range of target sites for suppression which will enable specific suppression of a single member of such a gene family.
The strategy described herein has applications for alleviating autosomal dominant diseases. Complete silencing of a disease allele may be difficult to achieve using antisense, ribozyme and triple helix approaches or any combination of these. However small quantities of mutant product may be tolerated in some autosomal dominant disorders. In others a significant reduction in the proportion of mutant to normal product may result in an amelioration of disease symptoms. Hence this strategy may be applied to any autosomal dominantly inherited disease in man where the molecular basis of the disease has been established. This strategy will enable the same therapy to be used to treat a wide range of different disease mutations within the same gene. The development of strategies will be important to future gene therapies for some autosomal dominant diseases, the key to a general strategy being that it circumvents the need for a specific therapy for every dominant mutation in a given disease-causing gene. This is particularly relevant in some disorders, for example, rhodopsin linked autosomal dominant RP (adRP), in which to date about 100 different mutations in the rhodopsin gene have been observed in adRP patients. The costs of developing designer therapies for each individual mutation which may be present in some cases in a single patient are prohibitive at present. Hence strategies such as this using a more universally applicable approach for therapy will be required.
This strategy may be applied in gene therapy approaches for biologically important polygenic disorders affecting large proportions of the world""s populations such as age related macular degeneration (ARMD) glaucoma, manic depression, cancers having a familial component and indeed many others. Polygenic diseases require the inheritance of more than one mutation (component) to give rise to the disease phenotype. Notably an amelioration in disease symptoms may require reduction in the presence of only one of these components, that is, suppression of one of the genotypes which, together with others, leads to the disease phenotype, may be sufficient to prevent or ameliorate symptoms of the disease. In some cases the suppression of more than one component giving rise to the disease pathology may be required to obtain an amelioration in disease symptoms. The strategy described here may be applied broadly to possible future interventive therapies in common polygenic diseases to suppress a particular genotype(s) and thereby suppress the disease phenotype.
In the present invention suppression effectors are designed specifically to target the non-coding regions of genes, for example, the 5xe2x80x2 and 3xe2x80x2 UTRs. This provides sequence specificity for gene suppression. In addition it provides greater flexibility in the choice of target sequence for suppression in contrast to suppression strategies directed towards single disease mutations. Furthermore it allows suppression effectors to target non-coding sequences 5xe2x80x2 or 3xe2x80x2 of the coding region thereby allowing the possibility of including the ATG start site in the target site for suppression and hence presenting an opportunity for suppression at the level of translation or inducing instability in RNA by, for example, cleavage of the RNA before the polyA tail.
Notably the invention has the advantage that the same suppression strategy when directed to the 5xe2x80x2 and 3xe2x80x2 non-coding sequences could be used to suppress, in principle, any mutation in a given gene. This is particularly relevant when large numbers of mutations within a single gene cause a disease pathology. Suppression targeted to non-coding sequences allows, when necessary, the introduction of a replacement gene(s) with the same or similar coding sequences to provide the normal gene product. The replacement gene can be designed to have altered non-coding sequences and hence can escape suppression as it does not contain the target site(s) for suppression. The same replacement gene could in principle be used in conjunction with the suppression of any disease mutation in a given gene. In the case of suppression of an individual member of a gene family, the non-coding regions typically show lower levels of homology between family members thereby providing more flexibility and specificity in the choice of target sites for suppression. In relation to this aspect of the invention, the use of intronic sequences for suppression of an individual member of a family of genes has been described in a previous invention (REF: WO 92/07071). However the use of 5xe2x80x2 and 3xe2x80x2 non-coding sequences as targets for suppression holds the advantage that these sequences are present not only in precursor messenger RNAs. but also in mature messenger RNAs, thereby enabling suppressors to target all forms of RNA. In contrast, intronic sequences are spliced out of mature RNAS.
In summary the invention can involve gene suppression and replacement such that the replacement gene cannot be suppressed. Both the same suppression and replacement steps can be used for many and in some cases all of the disease mutations identified in a given gene. Therefore the invention enables the same approach to be used to suppress a wide range of mutations within the same gene. Suppression and replacement can be undertaken in conjunction with each other or separately.